1. Field of the Invention
The present invention relates to a method of assay of micro-organisms, especially small wall-less bacteria such as mycoplasmas, in cell cultures.
2. Description of the Related Art
Mycoplasma are a large and widespread group of prokaryotes. They are essentially bacteria with the smallest known genomes and are found amongst members of mollicutes. This class presently comprises six eubacterial genera Acholeplasma, Anaeroplasma, Asteroplasma, Mycoplasma, Spiroplasma and Ureaplasma.
The Mycoplasmas and Ureaplasmas are parasites in different vertebrates, from which they obtain their essential compounds such as fatty acids, amino acids, precursors for nucleic acid synthesis and cholesterol. Only Acholeplasma and Asteroplasma do not require cholesterol for growth.
These flexible, pleomorphic organisms can be as small as 200-300 nm in diameter and can achieve very high densities in cell cultures, (107-108 organisms/mL), without discernible pH changes or turbidity. Contamination rates are even higher for cell lines routinely grown in antibiotic-containing medium. There are currently more than 120 species in six genera, many of which are pathogenic. The vast majority of cell culture contaminants belong to only 6 species of human, bovine or porcine origin, namely Mycoplasma hyorhinis, Mycoplasma arginini, Mycoplasma salivarium, Mycoplasma orale, Mycoplasma fermentans and Acholeplasma laidlawii. 
Surveys and research studies have repeatedly shown that 15-50% or more of all cell lines are contaminated by mycoplasmas. They alter virtually every property and parameter measured in cell cultures, including hybridoma selection rates, protein and nucleic acid synthesis, immunogenicity, chromosomal breakage and production of virus and proteins such as interferons and monoclonal antibodies. As a result of this widespread problem, research and production of biological materials is often unknowingly done using mycoplasmal contaminated cell cultures. Thus, the validity and significance of research and the safety of the biologicals produced from contaminated cell cultures are jeopardised.
Because of their small size and lack of cell wall, mycoplasmas often pass through the 450 or even 200 nm filters used to “sterilise” cell culture media and sera, resulting in low levels of these organisms being accidentally introduced into cultures during routine feeding.
Tests for detecting mycoplasmas fall into two basic categories: direct culture with media; or indirect tests measuring specific biochemical markers or other characteristics associated with mycoplasmas.
Direct culture requires the use of one or more complex nutritionally enriched media and carefully controlled environmental conditions. Even then, some mycoplasmal strains are difficult to grow in culture without cells. Properly done, with appropriate positive and negative controls, direct culture testing offers the greatest security, but is rather slow, usually requiring up to 28 days for completion.
Indirect tests include DNA fluorochrome staining, DNA probes, PCR, ELISA, autoradiography, immunofluorescence and specific biochemical assays. While faster than direct culture methods, usually taking only 1-5 days to complete, indirect tests are not yet as sensitive and usually require higher levels of contamination (e.g. 104 or more organisms/mL) for detection.
The PCR-based kit of Stratagene is widely used, but is expensive, additional PCR chemicals are required and the running of PCRs requires technical expertise not always conveniently available. Also, competition between the internal control template DNA and the mycoplasma DNA frequently results in the control not being visible on the gel when the sample is contaminated with mycoplasma.
It is a problem to find an alternative rapid, indirect method which does not require PCR skills.